Adsorbent for cytokine, method of adsorptive removal, and apparatus adsorptive removal

ABSTRACT

The present invention provides an adsorbent for efficiently adsorbing and removing cytokine in liquid and a method for removing cytokine in liquid by the adsorbent. Also, the present invention provides an adsorber for removing cytokine in liquid. Specifically, an adsorbent for cytokine is obtained by immobilizing a compound having a log P value of at least 2.50 (P is the partition coefficient in an octanol-water system) on a water-insoluble carrier. By contacting a liquid containing cytokine with the adsorbent for cytokine, the cytokine in the liquid can be effectively adsorbed and removed. Furthermore, cytokine can be efficiently adsorbed and removed by an adsorber comprising a container having an inlet and an outlet for liquid and a means for preventing the adsorbent from flowing out of the container, which is filled with the adsorbent.

TECHNICAL FIELD

The present invention relates to an adsorbent for adsorbing and removingat least one cytokine selected from the group consisting ofinterleukin-4 (hereinafter referred to as IL-4), interleukin-10(hereinafter referred to as IL-10) and interleukin-13 (hereinafterreferred to as IL-13) from body fluids, a method for adsorbing andremoving the cytokine with the adsorbent and an adsorber for thecytokine.

BACKGROUND ART

Immunocytes produce various active substances when causing immuneresponse. Some are proteinous substances called cytokine, and play avery important role as biophylatic factors, which are closely involvedin immune inflammatory reactions specific or nonspecific to variousantigens. Although indispensable for maintenance of homeostasis of aliving body, cytokines are excessively produced in conditions such asinflammation and are involved in the pathogenesis and prolongation ofinflammation.

IL-4 is a substance which was reported to be a B cell activating factorin 1982 by Howard and Paul, and also by Vitetta et al. Thereafter, thissubstance was found to act on cells other than B cells and given thename of IL-4. IL-4 is a glycoprotein having a molecular weight of 20kDa. T cells and mast cells are known as IL-4 producing cells. As thefunctions of IL-4, there are many functions such as the activation of Bcells, induction of differentiation and growth of B cells, induction ofgrowth of T cells and differentiation of T cells to Th2 cells, inductionof thymocyte growth, induction of NK cell- and LAK cell-activation,eosinophilic anti-tumor activity, macrophage anti-inflammatory activity,induction of mast cell growth, enhancement of expressions offibroblast/vascular endotherial cell adhesion molecules and enhancementof hematopoietic cell colony formation.

IL-10 is a substance which was identified as a cytokine productioninhibitory factor in 1989 by Mosmann et al. Initially, the substance wascalled a cytokine production inhibitory factor but was renamed as IL-10because the substance was found to have various other activities aswell. IL-10 is a homodimer-type glycoprotein having a molecular weightof 35 to 40 kDa. As IL-10 producing cells, known are Th0/Th2 cells,activated T cells, monocytes/macrophages, activated B cells,melanocytes, and the like. The functions of IL-10 include suppression ofIFN-γ production in T cells, induction of T cell growth, induction of Bcell growth, enhancement of monocyte ADCC activity, and suppression ofmonokine production in monocytes/macrophages.

IL-13 is a substance which was cloned in 1993 by Minty et al. in Franceand Brown et al. in USA. When IL-13 gene is introduced into COS cells,the gene is expressed as a glycoprotein having a molecular weight of 17kDa. Activated T cells are known as IL-13 producing cells. As functionsof IL-13, there are suppression of inflammatory cytokine production inLPS-stimulated monocytes, prolongation of the survival time of monocytesand induction of expression of CD23 and MHC Class II on the surface ofmonocytes and B cells.

These cytokines, IL-4, IL-10 and IL-13, are called anti-inflammatorycytokine, and all have functions of anti-inflammatory activity incommon, such as macrophage anti-inflammatory activity, suppression ofmonokine production in monocytes/macrophages and suppression ofinflammatory cytokine production in LPS-stimulated monocytes.

Recently, there are reports that in the condition of excessivebiological reaction caused by invasion, which is classified under thenew concept of systemic inflammatory response syndrome (SIRS), proteincalled inflammatory cytokines such as interleukin-1 (hereinafterreferred to as IL-1), interleukin-6 (hereinafter referred to as IL-6),interleukin-8 (hereinafter referred to as IL-8) and tumor necrosisfactor-α (hereinafter referred to as TNF-α) are overproduced. Theactivities thereof have been reported to mainly cause systemicinflammatory reaction, followed by not only tissue disorders andmulti-organ insufficiency, but also death.

Anti-inflammatory cytokines have been considered to suppress the actionof these inflammatory cytokines. However, recently, in conditions whichare included in the concept of compensatory anti-inflammatory responsesyndrome (CARS), an excessive amount of anti-inflammatory cytokines isproduced and as a result, suppression of immune reaction occurs, wherebya condition of easily being infected is caused. Therefore, in the caseof infection such as septicemia, organopathy also occurs.

In any case, cytokines should work locally and increase in concentrationof cytokines in blood is undesirable for the human body. Therefore, amethod for removing these cytokines from blood is desired.

An adsorbent for removing IL-1, IL-6, IL-8 and TNF-α which are calledinflammatory cytokine has already been disclosed (Japanese UnexaminedPatent Publication Nos. 257115/1996 and 257398/1996). These publicationsdisclose that these inflammatory cytokines can be adsorbed and removedwith an adsorbent comprising a compound having a log P value of at least2.50, which is immobilized on a water-insoluble carrier.

In the present invention, anti-inflammatory cytokines, of which thephysiological activities with respect to inflammation are exactly theopposite of those of the above-mentioned inflammatory cytokines, havebeen found to be adsorbable and removable with an adsorbent comprising acompound having a log P value of at least 2.50, which is immobilized ona water-insoluble carrier.

The object of the present invention is to provide an adsorbent forefficiently adsorbing and removing at least one cytokine selected fromthe group consisting of IL-4, IL-10 and IL-13 in body fluids, a methodfor adsorbing and removing cytokine in a solution using the adsorbentand an adsorbe for the cytokine.

DISCLOSURE OF INVENTION

The present inventors have conducted intensive studies on adsorbentscapable of efficiently adsorbing and removing at least one cytokineselected from the group consisting of IL-4, IL-10 and IL-13 in bodyfluids. As a result, the inventors have found that an adsorbentcomprising a compound having a log P value of at least 2.50, which isimmobilized on a water-insoluble carrier, can efficiently adsorb andremove at least one cytokine selected from the group consisting of IL-4,IL-10 and IL-13 in body fluids and the present invention was achieved.

Namely, the present invention relates to an adsorbent for at least onecytokine selected from the group consisting of IL-4, IL-10 and IL-113,comprising a compound having a log P value of at least 2.50 (P is apartition coefficient in an octanol-water system), which is immobilizedon a water-insoluble carrier.

In a preferable embodiment, the water-insoluble carrier is awater-insoluble porous carrier.

Also, the present invention relates to a method for removing at leastone cytokine selected from the group consisting of IL-4, IL-10 and IL-13in a body fluid, which comprises contacting a body fluid with anadsorbent for at least one cytokine selected from the group consistingof IL-4, IL-10 and IL-13; the adsorbent comprising a compound having alog P value of at least 2.50 (P is a partition coefficient in anoctanol-water system), which is immobilized on a water-insolublecarrier.

Furthermore, the present invention relates to an adsorber for removingat least one cytokine selected from the group consisting of IL-4, IL-10and IL-13 comprising a container having an inlet and an outlet for afluid and a means for preventing an adsorbent from flowing out of thecontainer, which is filled with an adsorbent for at least one cytokineselected from the group consisting of IL-4, IL-10 and IL-13; theadsorbent comprising a compound having a log P value of at least 2.50 (Pis a partition coefficient in an octanol-water system), which isimmobilized on a water-insoluble carrier.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 is a schematic cross sectional view of one example of theadsorber for cytokine of the present invention. In FIG. 1, numeral 1indicates an inlet for liquid, numeral 2 indicates an outlet for liquid,numeral 3 indicates the adsorbent for cytokine of the present invention,numerals 4 and 5 indicate filters through which liquid and componentscontained in the liquid can pass but the adsorbent for cytokine cannotpass, numeral 6 indicates a column and numeral 7 indicates an adsorberfor cytokine.

FIG. 2 is a graph showing the results of examining the relationshipbetween flow rate and pressure loss using three kinds of materials.

BEST MODE FOR CARRING OUT THE INVENTION

Herein, “IL-4” refers to glycoprotein having molecular weight ofapproximately 20 kDa.

“IL-10” refers to homodimer-type glycoprotein having molecular weight ofapproximately 35 to 40 kDa.

“IL-13” refers to glycoprotein having molecular weight of approximately17 kDa.

“Body fluid” refers to blood, plasma, serum, ascites, lymph, synovia,fractions obtained therefrom and other liquid components obtained from aliving body.

The adsorbent of the present invention comprises a compound having a logP value of at least 2.50, which is immobilized on a water-insolublecarrier. The log P value is a parameter that indicates thehydrophobicity of a compound. A typical partition coefficient P in anoctanol-water system is calculated as described below. First, a compoundis dissolved in octanol (or water) and an equal amount of water (oroctanol) is added thereto. After shaking the mixture for 30 minutes in aGriffin flask shaker (made by Griffin & George Ltd.), the mixture iscentrifuged for 1 to 2 hours at 2,000 rpm. The concentration of thecompound in the octanol layer and the water layer is measured at roomtemperature under atmospheric pressure by various methods such as aspectroscopic method or GLC and the partition coefficient P is foundfrom the following equation.P=Coct/Cw

-   Coct: concentration of the compound in the octanol layer-   Cw: concentration of the compound in the water layer

The adsorbent of the present invention comprises a compound having a logP value of at least 2.50 found in the above manner, which is immobilizedon a water-insoluble carrier.

Until now, many researchers have measured the log P values of variouscompounds and the obtained values have been organized by C. Hansch etal. (cf. “PARTITION COEFFICIENTS AND THEIR USES”; Chemical Reviews,vol.71, page 525 (1971)).

With respect to compounds for which the value is unknown, the value (Σf)calculated using hydrophobic fragmental constant f can be referred to,which is disclosed in “THE HYDROPHOBIC FRAGMENTAL CONSTANT”, ElsevierSci. Pub. Com., Amsterdam, (1977) by R. F. Rekker. The hydrophobicfragmental constant is a value indicating the hydrophobicity of variousfragments determined by statistical analysis based on many found valuesof log P. The sum of the f values of respective fragments thatconstitute a compound has been reported to nearly correspond with thelog P value of the compound.

In searching for compounds effective for adsorbing cytokine selectedfrom IL-4, IL-10 and IL-13, a compound having a log P value of at least2.50 is immobilized to a water-insoluble carrier. As a result,adsorption ability is found to be extremely high when hexadecylamine(Σf=7.22) is immobilized on a water-insoluble carrier as the compound.From these results, adsorption of cytokine by the adsorbent of thepresent invention is considered to occur due to hydrophobic interactionbetween cytokine and an atomic group introduced onto the carrier byimmobilizing a compound having a log P value of at least 2.50.

In the present invention, the compound that is immobilized to thewater-insoluble carrier is not particularly limited as long as thecompound has a log P value of at least 2.50. However, in the case ofbonding the compound to the carrier by a chemical bonding method, partof the compound is often eliminated. When the eliminated groupcontributes largely to the hydrophobicity of the compound, that is whenthe hydrophobicity of the atomic group that is immobilized onto thecarrier becomes smaller than Σf=2.50 due to elimination, the compound isnot suitable as the compound used in the present invention, consideringthe purpose of the present invention. A typical example thereof is thecase where isopentyl benzoate (Σf=4.15) is immobilized on a carrierhaving hydroxyl group by transesterification. In such a case, the atomicgroup that is actually immobilized onto the carrier is C₆H₅—CO— and theΣf value of the atomic group is at most 1. Whether such a compound issuitable as the compound used in the present invention can be determinedby whether or not the log P value of a compound wherein the part ofeliminated group is substituted with hydrogen is at least 2.50, orwhether or not the hydorophobicity (Σf) of the atomic group immobilizedonto the carrier by elimination is at least 2.50.

Among compounds having a log P value of at least 2.50, preferable arecompounds having a functional group that can be utilized for binding tothe carrier such as unsaturated hydrocarbons, alcohols, amines, thiols,carboxylic acids and derivatives thereof, halides, aldehydes,hydrazides, isocyanates, compounds having an oxirane ring includingglycidyl ether and halogenated silane. Typical examples of thesecompounds are amines such as n-heptylamine, n-octylamine, decylamine,dodecylamine, hexadecylamine, octadecylamine, 2-aminooctene,naphthylamine, phenyl-n-propylamine and diphenylmethylamine; alcoholssuch as n-heptyl alcohol, n-octyl alcohol, dodecyl alcohol, hexadecylalcohol, 1-octene-3-ol, naphthol, diphenyl methanol and4-phenyl-2-butanol, and glycidyl ethers of these alcohols; carboxylicacids such as n-octanoic acid, nonanoic acid, 2-nonenoic acid, decanoicacid, dodecanoic acid, stearic acid, arachidonic acid, oleic acid,diphenylacetic acid and phenylpropionic acid and derivatives thereofincluding acid halides, esters and amides; halides such as octylchloride, octyl bromide, decyl chloride and dodecyl chloride; thiolssuch as octanethiol and dodecanethiol; halogenated silanes such asn-octyltrichlorosilane and octadecyltrichlorosilane; and aldehydes suchas n-octylaldehyde, n-caprinaldehyde and dodecylaldehyde.

Besides these, compounds having a log P value of at least 2.50 amongcompounds wherein a hydrogen atom of the hydrocarbon part of the abovecompounds are substituted with a substituent containing a heteroatomsuch as halogen, nitrogen, oxygen and sulfur, or another alkyl group andcompounds having a log P value of at least 2.50 that are shown in thetables of pages 555 to 613 of the above review by C. Hansch et al,“PARTITION COEFFICIENTS AND THEIR USES”, Chemical Reviews, vol.71, 525(1971) can be used. However, the compounds are not limited thereto inthe present invention.

These compounds may be used alone or any two or more kinds may be usedtogether. These compounds can also be used together with a compoundhaving a log P value of less than 2.50.

The water-insoluble carrier in the adsorbent of the present inventionrefers to a carrier that is solid under normal temperature and normalpressure and is insoluble in water. The water-insoluble carrier in thepresent invention may be in the form of particle, sheet, fiber or hollowfiber, but is not particularly limited. The size of the carrier is alsonot particularly limited.

For example, when the adsorbent of the present invention is in the formof particles, the average particle size is preferably at least 5 μm andat most 1000 μm. When the average particle size is less than 5 μm, inthe case that cells are contained in body fluid, sufficient spacethrough which the cells can pass may not be obtained. When the averageparticle size is more than 1000 μm, adsorption ability per volume maynot sufficiently be obtained. From the viewpoint that cells can passsmoothly, the average particle size is more preferably at least 25 μmand at most 1000 μm. Particularly, from the viewpoint that cells canpass more easily and adsorption ability is obtained, the averageparticle size is preferably at least 40 μm and at most 600 μm.Specifically, when the body fluid is blood, the average particle size ispreferably at least 200 μm and at most 1000 μm, from the viewpoint thatblood cells can pass with ease, and more preferably at least 200 μm andat most 600 μm, from the viewpoint that adsorption ability is obtained.Also, the particle distribution is preferably narrow, for the reasonthat pressure loss is not increased.

When the adsorbent of the present invention is in the form of hollowfiber, the inner diameter is preferably at least 1 μm and at most 500μm. When the inner diameter is smaller than 1 μm, in the case that cellsare contained in body fluid, the cells may not sufficiently passthrough. When the inner diameter is larger than 500 μm, adsorptionability per volume tends to be insufficient. From the viewpoint thatcells can pass through smoothly, the inner diameter is more preferablyat least 2 μm and at most 500 μm. From the viewpoints that cells canpass through more easily and adsorption ability is obtained, the innerdiameter is most preferably at least 5 μm and at most 200 μm.

Typical examples of the water-insoluble carrier used in the adsorbent ofthe present invention are inorganic carriers such as glass beads andsilica gel; organic carriers comprising synthetic polymers such ascrosslinked polyvinyl alcohol, cross-linked polyacrylate, cross-linkedpolyacrylamide and cross-linked polystyrene or polysaccharides such ascrystalline cellulose, crosslinked cellulose, crosslinked agarose andcrosslinked dextrin; and composite carriers obtained by combining theabove such as organic-organic carriers and organic-inorganic carriers.

Of these, hydrophilic carriers are preferable, as non-specificadsorption is relatively small and adsorption selectivity for cytokineis favorable. The hydrophilic carrier refers to a carrier wherein thecontact angle to water when the compound that constitutes the carrier isa flat plate is at most 60 degrees. Various methods for measuringcontact angle to water are known and as shown in a book by Ikeda,(Experimental Chemical Selection: Colloid Chemistry, Chapter 4,Thermodynamics of Interface, pages 75-104 (1986) published by Shokabo),the method of measuring by placing a water droplet on a flat-platecompound is most common. Typical examples of a compound wherein thecontact angle to water measured by this method is at most 60 degrees arecarriers comprising cellulose, polyvinyl alcohol, hydrolyzedethylene-vinyl acetate copolymer, polyacrylamide, polyacrylic acid,polymethacrylic acid, polymethyl methacrylate, polyethylene grafted withpolyacrylic acid, polyethylene grafted with polyacrylamide and glass.

These water-insoluble carriers more preferably have a large number ofmicropores of a suitable size, that is have a porous structure. Acarrier having a porous structure includes a carrier having space(macropores) that is formed by agglomeration of microspherical particleswhen the base polymer matrix forms one spherical particle byagglomeration of microspherical particles, a carrier having microporesbetween the agglomerate of nuclei in one microparticle that constitutesthe base polymer matrix and a carrier having micropores when a copolymerhaving a three-dimensional structure (polymer network) is swelled withan organic solvent having affinity therewith.

From the viewpoint of adsorption ability per unit volume of theadsorbent, the water-insoluble carrier having a porous structure ispreferably porous overall rather than porous only on the surface. Also,the pore volume and specific surface area are preferably large, as longas adsorption ability is not lost.

An example of a carrier satisfying these preferable conditions is porouscellulose gel. Porous cellulose gel has the following advantages:

-   (1) porous cellulose gel is not destroyed or does not generate fine    powder by operations such as stirring because the gel has relatively    high mechanical strength and is tough, can be flowed at a high flow    rate as the gel dos not consolidate when packed in a column even    when body fluid is poured at a high flow rate and has a micropore    structure that is unsusceptible to change by high pressure steam    sterilization;-   (2) porous cellulose gel is hydrophilic as the gel is constituted by    cellulose, contains a large number of hydroxyl groups that can be    used for bonding ligand and has low non-specific adsorption;-   (3) adsorption volume that is not inferior to soft get can be    obtained as porous cellulose gel has relatively high strength even    when pore volume is large; and-   (4) porous cellulose gel is safer than synthetic polymer gel.    Consequently, porous cellulose gel is one of the most suitable    carriers for the present invention. However, the carrier of the    present invention is not limited thereto. The above carriers may be    used alone or any two or more kinds can be used together.

These water-insoluble carriers having a porous structure preferably havethe feature that the substance to be adsorbed can enter the microporesin a relatively large probability but entrance of other proteins isprevented as much as possible. The substances to be adsorbed by theadsorbent of the present invention are IL-4, which is a protein having amolecular weight of approximately 20 kDa, IL-10, which is a proteinhaving a molecular weight of approximately 35 to 40 kDa, and IL-13,which is a protein having a molecular weight of approximately 17 kDa. Inorder to efficiently adsorb these proteins, the cytokine must be able toenter into the micropores in a relatively large probability, butentrance of other proteins is prevented as much as possible. As thestandard for molecular weight of a substance that can enter into themicropores, exclusion limit molecular weight is generally used. Theexclusion limit molecular weight refers to the molecular weight of themolecule having the smallest molecular weight, among molecules thatcannot enter into (are excluded from) the micropores in gel permeationchromatography, as described in books (for example, Hiroyuki Hatano andToshihiko Hanai, “Experimental High Performance Liquid Chromatography”,Kagaku Dojin). In general, the exclusion limit molecular weight ofglobular proteins, dextrans and polyethlene glycol has been eagerlystudied. With respect to the exclusion limit of the carrier used in thepresent invention, the values obtained using globular protein aresuitably used.

As a result of studying carriers of various exclusion limit molecularweight, the range of exclusion limit molecular weight of globularprotein suitable for adsorbing the cytokines has been found to be atleast 5,000 and at most 600,000. When a carrier having an exclusionlimit molecular weight of globular protein of less than 5,000 is used,the amount of adsorbed cytokines is small and practicability decreases.When a carrier having an exclusion limit molecular weight of globularprotein of more than 600,000 is used, the amount of protein (mainlyalbumin) other than the cytokines that is adsorbed increases andpracticability decreases from the viewpoint of selectivity. Accordingly,the exclusion limit molecular weight of globular protein of the carrierused in the present invention is preferably at least 5,000 and at most600,000. From the viewpoint that adsorption ability and selectivity canbe obtained more effectively, the exclusion limit molecular weight ismore preferably at least 6,000 and at most 400,000, most preferably atleast 10,000 and at most 300,000.

The carrier preferably has a functional group that can be used inimmobilization reaction of a ligand. Typical examples of the functionalgroup are hydroxyl group, amino group, aldehyde group, carboxyl group,thiol group, silanol group, amide group, epoxy group, halogen group,succinylimide group and acid anhydride group, but not limited thereto.

Both a hard carrier and a soft carrier can be used as the carrier of thepresent invention. When using the adsorbent for extracorporealcirculation, clogging must not occur when the adsorbent is packed intothe column and fluid is passed through and therefore, sufficientmechanical strength is required. Consequently, the carrier used in thepresent invention is preferably a hard carrier. Herein, hard carrierrefers to in the case of a granular gel, a carrier wherein therelationship between pressure loss AP and flow rate, when the gel isevenly packed in a cylindrical column and an aqueous fluid is passedthrough, is a linear relation up to 0.3 kg/cm², as described below inReference Example.

The adsorbent of the present invention is obtained by immobilizing acompound having a log P value of at least 2.50 on a water-insolubleporous carrier. Various known methods for immobilization can be usedwithout any particular restriction. However, when the adsorbent of thepresent invention is used for an extracorporeal circulation treatment,from the viewpoint of safety, suppressing desorption and elution ofligands as much as possible in sterilization or treatment is important.Therefore, immobilization is preferably conducted by covalent bonding.

The immobilized amount of the compound having a log P value of at least2.50 in the absorbent of the present invention is preferably at least 1μmol/g (wet weight) and at most 5000 μmol/g (wet weight). When theimmobilized amount is less than 1 μmol/g (wet weight), adsorption ofcytokine tends to be insufficient. When the immobilized amount is morethan 5000 μmol/g (wet weight), in that case that the fluid is blood,adhesion of platelets tends to occur. From the viewpoints of effectiveadsorption ability and little adhesion of platelets, the immobilizedamount of the compound is more preferably at least 5 μmol/g (wet weight)and at most 3000 μmol/g (wet weight).

There are various methods for adsorbing and removing the cytokine frombody fluid using the adsorbent of the present invention. The most simplemethod is the method of retrieving body fluid and storing in a bag,mixing the adsorbent thereto to adsorb and remove the cytokine and thenfiltering the adsorbent to obtain the body fluid from which the cytokinehas been removed. Another method is the method of filling a containerhaving an inlet and an outlet for body fluid, wherein a filter thatpasses body fluid but not the adsorbent is installed to the outlet, withthe adsorbent and then pouring body fluid. Both methods can be used, butthe latter method is suited for the adsorbent of the present invention,as operation is simple and the cytokine can be removed efficiently byon-line system from body fluid, especially blood, of a patient byincorporating the method into an extracorporeal circulation circuit.

In the extracorporeal circulation circuit, the adsorbent of the presentinvention can be used alone or together with other extracorporealcirculation therapy systems. An example of using together with anotheris artificial dialysis circuit and the adsorbent can be together withdialysis therapy.

Below, the adsorber for cytokine of the present invention using theabove cytokine adsorbent is explained based on FIG. 1, which isschematic cross sectional view of one example of the adsorber. In FIG.1, numeral 1 indicates an inlet for liquid, numeral 2 indicates anoutlet for liquid, numeral 3 indicates the adsorbent for cytokine of thepresent invention, numerals 4 and 5 indicate filters through whichliquid and components contained in the liquid can pass but the adsorbentfor cytokine cannot pass, numeral 6 indicates a column and numeral 7indicates an adsorber for cytokine. The adsorber for cytokine of thepresent invention is not limited to this example and any device can beused as long as the device has an inlet and an outlet for fluid and theadsorbent is packed in a container equipped with a means to prevent theadsorbent for cytokine from flowing out of the container.

Examples of the means for preventing the adsorbent from flowing out are,for instance, filters such as mesh, nonwoven fabric and cotton plug. Theshape, material and size of the container are not particularly limited,but the shape of the container is preferably cylindrical. The materialof the container is preferably a material having sterilizationresistance and specific examples are glass coated with silicone,polypropylene, polyvinyl chloride, polycarbonate, polysulfone andpolymethylpentene. The capacity of the container is preferably at least50 ml and at most 1500 ml, and the diameter is preferably at least 2 cmand at most 20 cm. When the capacity of the container is less than 50ml, the adsorbed amount is insufficient, whereas when the capacity ofthe container is more than 1500 ml, the extracorporeally circulatedamount becomes large, thus being unfavorable. When the diameter of thecontainer is less than 2 cm, the pressure loss becomes large as thelinear velocity is high, thus being unfavorable. When the diameter ofthe container is more than 20 cm, coagulation may occur as handling isdifficult and linear velocity is low, thus being unfavorable. From theviewpoints of an effective adsorption amount and excellent safety, thecontainer more preferably has capacity of at least 100 ml and at most800 ml and diameter of at least 3 cm and at most 15 cm, particularlypreferably capacity of at least 150 ml and at most 400 ml and diameterof at least 4 cm and at most 10 cm.

Hereinafter, the present invention is described in more detail by meansof Examples, but the present invention is not limited thereto.

REFERENCE EXAMPLE

A cylindrical glass column (inner diameter 9 mm, length 150 mm) equippedwith filters having a pore size of 15 μm at both ends was uniformlycharged with each of agarose material (Biogel A-5m available fromBio-Rad Laboratories, particle size 50 to 100 mesh), vinyl polymermaterial (TOYOPEARL HW-65 available from TOSOH Corporation, particlesize 50 to 100 μm) and cellulose material (CELLULOFINE GC-700m availablefrom Chisso Corporation, particle size 45 to 105 μm). Then, water waspassed through by a peristatic pump. The relationship between flow rateand pressure loss AP was found. The results are shown in FIG. 2.

As shown in FIG. 2, the flow rate of TOYOPEARL HW-65 and CELLULOFINEGC-700m increase almost in proportion to increase in pressure. On theother hand, Biogel A-5m consolidates and the flow rate does not increaseeven when the pressure is increased. In the present invention, amaterial wherein the relationship between pressure loss AP and flow rateis a linear relation up to 0.3 kg/cm², as in the case of the former, isreferred to as a hard material.

Example 1

Water was added to 170 ml of porous cellulose gel CELLULOFINE GC-200m(available from Chisso Corporation, particle size: 45 to 105 μm,exclusion limit molecular weight for globular protein: 140,000) so thatthe total amount became 340 ml. Then, 90 ml of a 2M aqueous solution ofsodium hydroxide was added thereto, and the temperature was adjusted to40° C. To the mixture was added 31 ml of epichlorohydrin and reactionwas carried out while stirring at 40° C. for 2 hours. After the reactionwas completed, the gel was thoroughly washed with water to obtain anepoxidized gel.

To 10 ml of the epoxidized gel was added 200 mg of n-hexadecylamine(Σf=7.22) and the mixture was left still to react in ethanol at 45° C.for 6 days to immobilize. After the reaction was completed, the gel wasthoroughly washed with ethanol and then with water to obtainn-hexadecylamine-immobilized gel (immobilized amount of hexadecylamine:45 μmol/g (wet weight)).

To 0.5 ml of the immobilized gel was added 3 ml of cytokine-containingserum of a healthy volunteer prepared by adding human gene recombinantIL-4, IL-10 and IL-13 (all available from R & D systems) to serum of ahealthy volunteer (available from Dainippon Pharmaceutical Co., Ltd.).The mixture was incubated at 37° C. for 2 hours. After incubation, theconcentration of each cytokine in the supernatant was measured using ahuman IL-4 measurement kit, a human IL-10 measurement kit and a humanIL-13 measurement kit (all available from Biosource InternationalCorporation). The results are shown in Table 1.

Example 2

n-Octylamine-immobilized gel (immobilized amount of n-octylamine: 43μmol/g (wet weight)) was obtained in the same manner as in Example 1,except that n-octylamine (log P=2.90) was used instead ofn-hexadecylamine and 50 (v/v) % aqueous solution of ethanol was used asthe medium for immobilization instead of ethanol. Adsorption test wascarried out in the same manner as in Example 1 using this immobilizedgel and the concentration of each cytokine was measured. The results areshown in Table 1.

Comparative Example 1

n-Hexylamine-immobilized gel (immobilized amount of n-hexylamine: 46μmol/g (wet weight)) was obtained in the same manner as in Example 2except that n-hexylamine (log P=2.06) was used instead of n-octylamine.Adsorption test was carried out in the same manner as in Example 1 usingthis immobilized gel and the concentration of each cytokine wasmeasured. The results are shown in Table 1.

Comparative Example 2

n-Butylamine-immobilized gel (immobilized amount of n-butylamine: 48μmol/g (wet weight)) was obtained in the same manner as in Example 2except that n-butylamine (log P=0.97) was used instead of n-octylamine.Adsorption test was carried out in the same manner as in Example 1 usingthis immobilized gel and the concentration of each cytokine wasmeasured. The results are shown in Table 1.

Comparative Example 3

Adsorption test was carried out in the same manner as in Example 1 usingCELLULOFINE GC-200m, on which a ligand was not immobilized, and theconcentration of each cytokine was measured. The results are shown inTable 1. TABLE 1 Cytokine Concentration (pg/ml) IL-4 IL-10 IL-13 Ex. 1 965 150 Ex. 2 15 80 265 Com. Ex. 1 240 385 2000 Com. Ex. 2 240 390 2100Com. Ex. 3 250 400 2300

As can be seen from Table 1, the concentration of each cytokine in thesupernatant is significantly decreased in Examples 1 and 2 compared toComparative Examples 1, 2 and 3.

Example 3

Cellulose acetate was dissolved in a mixed solvent of dimethylsulfoxideand propylene glycol. The solution was dropped by the method describedin JP-A-63-117039 (vibration method) and coagulated to obtain sphericalhydrogel particles of cellulose acetate. The hydrogel particles weremixed with an aqueous solution of sodium hydroxide and hydrolysisreaction was conducted to obtain hydrogel particles of cellulose(average particle size: 460 μm, exclusion limit moleuclar weight forglobular protein: 50,000). The hydrogel particles were reacted withepichlorohydrin in the same manner as in Example 1 and n-hexadecylaminewas immobilized onto the particles to obtainn-hexadecylamine-immobilized particles (immobilized amount ofn-hexadecylamine: 35 μmol/g (wet weight)). Adsorption test was carriedout in the same manner as in Example 1 using the immobilized particlesand the concentration of each cytokine was measured. The results areshown in Table 2.

Example 4

n-Octylamine-immobilized particles (immobilized amount of n-octylamine:33 μmol/g (wet weight)) were obtained in the same manner as in Example 3except that n-octylamine was used instead of n-hexadecylamine and that50 (v/v) % aqueous solution of ethanol was used as the medium forimmobilization reaction instead of ethanol. Adsorption test was carriedout in the same manner as described in Example 1 using the immobilizedparticles and the concentration of each cytokine was measured. Theresults are shown in Table 2.

Comparative Example 4

Adsorption test was carried out in the same manner as in Example 1 usingthe hydrogel particles of cellulose on which a ligand was notimmobilized prepared in Example 3 and the concentration of each cytokinewas measured. The results are shown in Table 2. TABLE 2 CytokineConcentration (pg/ml) IL-4 IL-10 IL-13 Ex. 3 10 70 170 Ex. 4 20 80 270Com. Ex. 4 260 390 2300

As can be seen from Table 2, the concentration of each cytokine in thesupernatant is significantly decreased in Examples 3 and 4 compared toComparative Example 4.

INDUSTRIAL APPLICABILITY

By using the adsorbent obtained by immobilizing a compound having a logP value of at least 2.50 onto a water-insoluble carrier according to themethod of the present invention, at least one cytokine selected from thegroup consisting of IL-4, IL-10 and IL-13 can be efficiently adsorbedand removed.

1. An adsorbent for adsorbing at least one cytokine selected from thegroup consisting of interleukin-4, interleukin-10 and interleukin-13,comprising a compound having a log P value of at least 2.50 (P is apartition coefficient in an octanol-water system), which is immobilizedon a water-insoluble carrier.
 2. The adsorbent of claim 1, wherein saidwater-insoluble carrier is a water-insoluble porous carrier.
 3. A methodfor removing at least one cytokine selected from the group consisting ofinterleukin-4, interleukin-10 and interleukin-13 in a body fluid, whichcomprises contacting a body fluid with an adsorbent for adsorbing atleast one cytokine selected from the consisting of interleukin-4,interleukin-10 and interleulkin-13; said adsorbent comprising a compoundhaving a log P value of at least 2.50 (P is a partition coefficient inan octanol-water system), which is immobilized on a water-insolublecarrier.
 4. An adsorber for adsorbing at least one cytokine selectedfrom the group consisting of interleukin-4, interleukin-10 andinterleulkin-13, comprising a container having an inlet and an outletfor a body fluid and a means for preventing an adsorbent from flowingout of said container, which is filled with an adsorbent for adsorbingat least one cytokine selected from the consisting of interleukin-4,interleukin-10 and interleukin-13; said adsorbent comprising a compoundhaving a log P value of at least 2.50 (P is a partition coefficient inan octanol-water system), which is immobilized on a water-insolublecarrier.
 5. Use of an adsorbent comprising a compound having a log Pvalue of at least 2.50 (P is a partition coefficient in an octanol-watersystem), which is immobilized on a water-insoluble carrier, foradsorbing and removing from a body liquid at least one cytokine selectedfrom the group consisting of interleukin-4, interleukin-10 andinterleukin-13.